Stress induces cells to develop Tolerance against subsequent exposure to a diverse range of toxic insults. There are two phases of Tolerance. One phase develops quickly and requires no protein synthesis (protein synthesis independent Tolerance: PSIT). Stress-induced protein synthesis is requisite for the slower developing, more protracted second phase (protein synthesis dependent Tolerance: PSDT). The central hypothesis is that heat shock proteins (HSPs) confer PSIT and PSDT stress protection. We proposed a model in which stress-denatured and aggregated proteins induce cells to develop PSIT and/or PSDT. PSIT develops more quickly and requires constitutively expressed HSP-27 and/or alphaB-crystallin to maintain stress-denatured and aggregated proteins in a folding competent state, making the proteins more accessible to disaggregation by the unfolding/refolding chaperone activity of HSC-70 and/or HSP-70. If PDT is also induced, PSIT protects cells while additional HSP-27, alphaB-crystallin and HSP-70/HSC-70 are synthesized. Up-regulated HSPs then provide protracted PSDT protection after PSIT decays. In both PSIT and PSDT, HSC70 and/or HSP 70 ultimately disaggregate large protein aggregates into smaller ones. Proteins in the smaller aggregates are either returned to their native state by continued HSC-70 or HSP-70 unfolding/refolding, or more efficiently proteolyzed. The specific aims and experimental design will test critical aspects pf the model by determining how PSIT and PSDT are induced, and how HSP-27 and alphaB-crystallin confer PSIT and PSDT stress protection. This will include determining how stress-induced: phosphorylation, changes in oligomer size, and changes in cellular distribution of HSP-27 and alphaB- crystallin influence their stress protection. Experiments will also establish if HSP-27 and alphaB-crystallin function in a cooperative manner with HSC-70 and HSP-70 to provide stress protection, and discover the conditions that determine whether denatured proteins are disaggregated into a soluble state of proteolyzed. Tolerance provides protection against many clinically relevant stresses, e.g., ischemia, and denatured and abnormal proteins are involved in many human diseases including, Alzheimer's disease. Thus, understanding how HSPs function in PSIT and PSDT to protect cells against cytotoxic stresses and to ameliorate the cytotoxicity of denatured and aggregated proteins may lead to new and effective clinical applications for stress Tolerance.